Cyclic hexapeptide somatostatin analogs

ABSTRACT

Somatostatin analogs are prepared wherein a cyclic hexapeptide contains a secondary amino acid which replaces seven of the ring amino acids of somatostatin. The cyclic hexapeptides are easier to synthesize, have a longer duration of activity, and many have a greater level of activity than somatostatin. The compounds have the properties of inhibiting the release of glucagon, growth hormone and insulin. Certain of the compounds also are capable of inhibiting gastric acid secretions. The compounds are particularly useful in the treatment of acromegaly, diabetes, diabetic retinopathy and peptic ulcers. These cyclic hexapeptides are prepared by the solid phase method.

BACKGROUND OF THE INVENTION

Somatostatin is a tetradecapeptide incorporating a cyclic dodecapeptide, having the structure: ##STR1## and has the properties of inhibiting the release of growth hormone, inhibiting the release of insulin and glucagon and reducing gastric secretions. Somatostatin itself has a short duration of action because it is inactivated, inter alia, by aminopeptidases and carboxypeptidases present in vivo. This problem of the short duration of action has been partially solved in the prior art by preparing derivatives of somatostatin which have low solubility, thus attaining a slow release on subcutaneous injection. Once dissolved, however, the derivatives are no more stable to inactivation by aminopeptidases and carboxypeptidases than somatostatin itself.

SUMMARY OF THE INVENTION

The present invention provides for cyclic hexapeptides which are derivatives of somatostatin in which, inter alia, seven of the ring amino acids are replaced by a secondary amino acid, and both of the exocyclic amino acids are removed. Further substitution and reaction of the remaining amino acids is also described. The cyclic hexapeptides inhibit the release of glucagon, growth hormones and insulin, and inhibit the release of gastric acid secretions. Specifically the compounds may preferentially inhibit the release of growth hormones without affecting the level of gastric secretions or without affecting the level of gastric secretions, insulin and glucagon, or the compounds may inhibit the release of gastric acid secretions. Thus, the compounds have a more selective biological activity than somatostatin. The cyclic hexapeptide structures of the instant compounds also have a longer duration of activity than somatostatin. As such the instant cyclic hexapeptides are useful for the treatment of acromegaly, diabetes, diabetic retinopathy and peptic ulcers.

Thus, it is an object of the present invention to describe the cyclic hexapeptide somatostatin analogs. A further object is to describe procedures for the preparation of such cyclic hexapeptides. A still further object is to describe the use of such compounds in the treatment of acromegaly, diabetic retinopathy and peptic ulcers. Further objects will become apparent from reading the following description.

The compounds are particularly effective when administered to Type I (insulin dependent) diabetics. The compounds have the property of preventing large increases in blood sugar which normally occur in such patients following the ingestion of a meal.

DESCRIPTION OF THE INVENTION

The compounds of the instant invention are best realized in the following structural formulae: ##STR2## wherein X is (CH₂)_(n) wherein n is 0, 1, or 2 or sulfur;

Y is (CH₂)_(m) wherein m is 0, 1 or 2 or sulfur such that the sulfur may be in any position along the chain;

R₁ and R₂ are independently loweralkyl, benzyl, substituted benzyl where the substituent may be one or two of loweralkyl, halogen, hydroxy, amino, nitro or loweralkoxy; and loweralkyl substituted with a 5- or 6- membered heterocyclic ring;

R₃ is 3-indolylmethyl or substituted 3-indolylmethyl wherein the substituent may be loweralkyl, loweralkoxy, or halogen;

R₄ is loweralkyl, hydroxyloweralkyl, benzyl, carboxyloweralkyl, aminoloweralkyl or substituted benzyl wherein the substituent may be loweralkyl, loweralkoxy, hydroxy, halogen, amino or nitro;

R₅ is loweralkyl, benzyl, or substituted benzyl wherein the substituent is loweralkyl, loweralkoxy, hydroxy, halogen amino or nitro; and

R₆ and R₇ are independently hydrogen or straight chain lower alkyl provided at least one of R₆ and R₇ is an alkyl.

The term "loweralkyl" when used in the instant application is intended to represent those alkyl groups either straight or branched chain, which have from 1-5 carbon atoms. Examples of such alkyl groups are methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl, pentyl and the like.

The term "loweralkoxy" is intended to include those alkoxy groups of from 1-5 carbon atoms, in either a straight or branched chain. Examples of such alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentoxy and the like.

The term "halogen" or "halo" is intended to include fluorine, chlorine, bromine and iodine.

The term "5- or 6-membered heterocyclic ring" is intended to include those 5- and 6-membered heterocycles with 1- or 2-heteroatoms selected from oxygen, nitrogen and sulfur. Exemplary of such heterocycles is imidazole, furan, thiazole, pyrazole, pyridine and the like.

In the instant compounds there are several asymmetric centers which will lead to the existence of optical isomers for such compounds. In the instant invention, for each of the asymmetric centers of the various amino acids which make up the instant cyclic hexapeptides, both the D and L configurations are intended to be encompassed.

It will be appreciated by those skilled in the art that when R₁ and R₂ are benzyl, R₃ is indolylmethyl, Y is methylene, and R₄ is 1-hydroxyethyl, the 7,8,9,10 and 11 amino acids of somatostatin (-Phe-Trp-Lys-Thr-Phe-) are represented, and the secondary amino acid, represented by N-methyl alanine when R₅ is methyl, in structure I, and by proline when X is methylene in structure II, has taken the place of the remainder of the somatostatin amino acids while R₆ and R₇ allow for N-alkylation of the 9-position lysine and 11-position phenylalanine. Thus, using the above definitions of the substituent groups, the following representative cyclic hexapeptide analogs of somatostatin are formed in structure I; ##STR3##

The preferred embodiments of the cyclic hexapeptides of this invention are realized in the foregoing structural formula I wherein Y is (CH₂)_(n) and n is 1;

R₁ and R₂ are as defined above;

R₃ is 3-indolylmethyl or substituted indolylmethyl wherein the substituent is methoxy or fluoro;

R₄ is methyl, ethyl, isopropyl, hydroxymethyl or hydroxyethyl;

R₅ is methyl or benzyl; and one or both of R₆ and R₇ are methyl.

Further preferred embodiments are realized when Y is methylene;

R₁ and R₂ are as defined above;

R₃ is 3-indolylmethyl;

R₄ is hydroxy ethyl;

R₅ is methyl or benzyl; and one or both of R₆ and R₇ is methyl.

The preferred R₁ and R₂ groups are loweralkyl, benzyl or substituted benzyl where the substituent is loweralkyl, halogen, hydroxy, amino, nitro or alkoxy.

Included within these preferred compounds are:

Cyclo-(N-Me-Ala-Tyr-D-Trp-Lys-Thr-N-Me-Phe)

Cyclo-(N-Me-Ala-Phe-D-Trp-Lys-Thr-N-Me-Phe)

Cyclo-(N-Me-Ala-Phe-L-Trp-Lys-Thr-N-Me-Phe)

Cyclo-(N-Me-Phe-Tyr-D-Trp-Lys-Val-N-Me-Ala)

Cyclo-(N-Me-Phe-Phe-D-Trp-Lys-Thr-N-Me-Ala)

Cyclo-(N-Me-Phe-His-D-Trp-Lys-Ser-N-Me-Phe)

Cyclo-(N-Me-Ala-Tyr-D-Trp-Lys-Val-N-Me-Phe)

Cyclo-(N-Me-Ala-Tyr-D-Trp-N-Me-Lys-Thr-Phe

Cyclo-(N-Me-Ala-Phe-D-Trp-N-Me-Lys-Thr-Phe)

Cyclo-(N-Me-Ala-Phe-L-Trp-N-Me-Lys-Thr-Phe)

Cyclo-(N-Me-Ala-Phe-D-Trp-N-Me-D-Lys-Thr-Phe)

Cyclo-(N-Me-Ala-Tyr-D-Trp-N-Me-D-Lys-Val-Phe)

Cyclo-(N-Me-Ala-Tyr-D-Trp-N-Me-Lys-Val-Phe)

Cyclo-(N-Me-Phe-His-D-Trp-N-Me-Lys-Ser-Ala)

Cyclo-(N-Me-Ala-Tyr-D-Trp-N-Me-Lys-Thr-N-Me-Phe

Cyclo-(N-Me-Ala-Phe-D-Trp-N-Me-Lys-Thr-N-Me-Phe)

Cyclo-(N-Me-Ala-Phe-L-Trp-N-Me-Lys-Thr-N-Me-Phe)

Cyclo-(N-Me-Ala-Phe-D-Trp-N-Me-D-Lys-Thr-N-Me-Phe)

Cyclo-(N-Me-Ala-Tyr-D-Trp-N-Me-D-Lys-Val-N-Me-Phe)

Cyclo-(N-Me-Phe-His-D-Trp-N-Me-Lys-Ser-N-Me-Ala)

Cyclo-(N-Me-Ala-Tyr-D-Trp-N-Me-Lys-Val-N-Me-Phe)

Using the above definitions of the substituent groups, the following representative cyclic hexapeptide analog of somatostatin is formed in structure II: ##STR4##

The preferred embodiments of the cyclic hexapeptides of this invention are realized in the foregoing structural formula II where:

X and Y are (CH₂)_(n) and n is 1;

R₁ and R₂ are as defined above;

R₃ is 3-indolylmethyl or substituted indolylmethyl wherein the substituent is methoxy or fluoro;

R₄ is methyl, ethyl, isopropyl, hydroxy methyl or hydroxy ethyl; and one or both of R₆ and R₇ is methyl.

Further preferred embodiments are realized when X and Y are methylene;

R₁ and R₂ are as defined above;

R₃ is 3-indolylmethyl;

R₄ is hydroxy ethyl; and

one or both of R₆ and R₇ is methyl

The preferred R₁ and R₂ groups are loweralkyl, benzyl or substituted benzyl where the substituent is loweralkyl, halogen, hydroxy, amino, nitro or alkoxy.

Included within these preferred compounds are:

Cyclo-(Pro-Tyr-D-Trp-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-D-Trp-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-L-Trp-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Tyr-D-Trp-Lys-Val-N-Me-Phe)

Cyclo-(Pro-Phe-D-5-F-Trp-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-L-5-F-Trp-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-D-Trp-Lys-Ser-N-Me-Phe)

Cyclo-(Pro-Tyr-D-Trp-N-Me-Lys-Thr-Phe)

Cyclo-(Pro-Phe-D-Trp-N-Me-Lys-Thr-Phe)

Cyclo-(Pro-Phe-L-Trp-N-Me-Lys-Thr-Phe)

Cyclo-(Pro-Phe-D-Trp-N-Me-D-Lys-Thr-Phe)

Cyclo-(Pro-Phe-D-5-F-Trp-N-Me-Lys-Thr-Phe)

Cyclo-(Pro-Phe-L-5-F-Trp-N-Me-Lys-Thr-Phe)

Cyclo-(Pro-Tyr-D-Trp-N-Me-Lys-Val-Phe)

Cyclo-(Pro-Tyr-D-Trp-N-Me-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-D-Trp-N-Me-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-L-Trp-N-Me-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-D-Trp-N-Me-D-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-D-5-F-Trp-N-Me-Lys-Thr-N-Me-Phe)

Cyclo-(Pro-Phe-L-5-F-Trp-N-Me-Lys-Thr N-Me-Phe)

Cyclo-(Pro-Tyr-D-Trp-N-Me-Lys-Val-N-Me-Phe)

In the instant application several abbreviated designations are used for the amino acid components, certain preferred protecting groups, reagents and solvents. The meanings of such abbreviated designations are given in Table I.

                  TABLE I                                                          ______________________________________                                         Abbreviated                                                                    Designation                                                                    ______________________________________                                                        Amino Acid                                                      Lys            L-lysine                                                        Phe            L-phenylalanine                                                 Trp            L-tryptophan                                                    D-Trp          D-tryptophan                                                    Thr            L-threonine                                                     Aha            7-aminoheptanoic acid                                           Tyr            L-tyrosine                                                      Val            L-valine                                                        Abu            L-α-aminobutyric acid                                     Ser            L-serine                                                        Asn            L-asparagine                                                    Pro            L-proline                                                       Asu            D- or L-aminosuberic acid                                       Cys            L-cysteine                                                      AChxAla        amino cyclohexylalanine                                         AmPhe          aminomethyl phenylalanine                                       His            L-Histidine                                                                    Protecting                                                                     Groups                                                          INOC           isonicotinyloxycarbonyl                                         BOC            tert-butyloxycarbonyl                                           OMe            methyl ester                                                    Bu             tert-butyl                                                      CBZ            benzyloxycarbonyl                                               Bzl            benzyl                                                          2-Cl--CBZ      2-chlorobenzyloxycarbonyl                                       Acm            acetamidomethyl                                                 Me             methyl                                                          FMOC           9-fluorenylmethyloxycarbonyl                                                   Activating                                                                     Groups                                                          ONp            p-nitrophenyl ester                                             HSE            N--hydroxysuccinimide                                           ester                                                                          HBT            1-hydroxybenzotriazole                                                         Condensing                                                                     Agents                                                          DCCI           dicyclohexylcarbodiimide                                                       Reagents                                                        TFA            trifluoroacetic acid                                            TEA            triethylamine                                                   DIPEA          diisopropylethylamine                                                          Solvents                                                        EPAW           ethyl acetate-pyridine-                                                        acetic acid-water                                               BAW            butanol-acetic acid-water                                       CMW            chloroform-methanol-water                                       DMF            dimethylformamide                                               THF            tetrahydrofuran                                                 ______________________________________                                    

In accordance with the present invention, the novel cyclic hexapeptide somatostatin analogs are prepared by cyclizing corresponding linear peptides. The linear peptides are prepared by using the solid phase sequential synthesis technique. Accordingly, the process for preparing the cyclic hexapeptide somatostatin analogs of the present invention comprises (a) preparing a corresponding blocked linear peptide attached to a solid phase resin; (b) selectively deblocking the N-terminal amine group; (c) removing the linear peptide from the resin; (d) treating the linear peptide with a cyclizing agent to obtain the cyclic hexapeptide through the formation of an amide bond; (e) removing any side chain blocking groups.

When the linear peptide is prepared on the resin, it is generally not critical which amino acid is selected to be at the C-terminal position provided only that the sequence of amino acids in the linear peptide corresponds to that in the desired somatostatin analog. Once a linear peptide has been cyclized one can no longer determine which amino acid was at the C-terminus of the linear peptide.

While generally the selection of the first amino acid to start the chain is not critical, since the linear peptide will be cyclized, there may be other factors which may prefer one starting amino acid over another. For example, D-Trp can react with t-butyl carbonium ions which are formed when BOC groups are removed. Thus, selection of a reaction sequence which places D-Trp at the N-terminal end of the linear peptide will cause D-Trp to be added last, and thus it will have the least exposure to t-butyl carbonium ions. This type of selection may not always be possible, such as where there are two indole containing moieties in the peptide. However, such reaction sensitivities should be considered when planning a peptide reaction sequence.

The synthesis of the linear peptides by the solid phase technique is conducted in a stepwise manner on chloromethylated resin. The resin is composed of fine beads (20-70 microns in diameter) of a synthetic resin prepared by copolymerization of styrene with 1-2 percent divinylbenzene. The benzene rings in the resin are chloromethylated in a Friedel-Crafts reaction with chloromethyl methyl ether and stannic chloride. The Friedel-Crafts reaction is continued until the resin contains 0.5 to 5 mmoles of chlorine per gram of resin.

The amino acid selected to be the C-terminal amino acid of the linear peptide is converted to its amino protected derivative. The carboxyl group of the selected C-terminal amino acid is bound covalently to the insoluble polymeric resin support, as for example, as the carboxylic ester of the resin-bonded benzyl chloride present in chloromethyl-substituted polystyrene-divinylbenzene resin. After the amino protecting group is removed, the amino protected derivative of the next amino acid in the sequence is added along with a coupling agent, such as dicyclohexylcarbodiimide. The amino acid reactant may be employed in the form of a carboxyl-activated amino acid such as the ONp ester, an amino acid azide, and the like. Deprotection and addition of successive amino acids is performed until the desired linear peptide is formed.

The selection of protecting groups is, in part, dictated by particular coupling conditions, in part by the amino acid and peptide components involved in the reaction.

Amino-protecting groups ordinarily employed include those which are well known in the art, for example, urethane protecting substituents such as benzyloxycarbonyl (carbobenzoxy), p-methoxycarbobenzoxy, p-nitrocarbobenzoxy, t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, and the like. It is preferred to utilize t-butyloxycarbonyl (BOC) for protecting the α-amino group in the amino acids undergoing reaction at the carboxyl end of said amino acid. The BOC protecting group is readily removed following such coupling reaction and prior to the subsequent step by the relatively mild action of acids (i.e., trifluoroacetic acid, or hydrogen chloride in ethyl acetate). Another preferred protecting group for the α-amino group is FMOC, which is readily removed following coupling with diisopropylamine in DMF or piperidine.

The OH group of Thr and Ser can be protected by the Bzl group and the ω-amino group of Lys,N-Me-Lys, AChxAla and AmPhe can be protected by the INOC group or the 2-chlorobenzyloxycarbonyl (2-Cl-CBZ) group. In the case of Lys,N-Me-Lys, AChxAla and AmPhe, it is preferred to protect the ω-amino group with 2-Cl-CBZ group as this group is removed simultaneously with the Bzl groups by treatment with HF after the linear peptide has been cyclized. The INOC group is not removed by HF and requires an additional treatment with Zn. Neither group is affected by TFA, used for removing BOC protecting groups. After the linear peptide is cyclized, the protective groups, such as 2-Cl-CBZ and Bzl, are removed by treatment with HF.

After the linear peptide has been formed on the solid phase resin, it may be removed from the resin by a variety of methods which are well known in the art. For example, the peptide may be cleaved from the resin with hydrazine and thus directly form the peptide hydrazide which may be subsequently cyclized via the azide to the desired cyclic peptide. The hydrazide is converted to the corresponding azide by reaction with a reagent which furnishes nitrous acid in situ. Suitable reagents for this purpose include a lower alkyl nitrite (e.g., t-butyl nitrite, isoamyl nitrite) or an alkali metal nitrite salt (e.g., sodium nitrite, potassium nitrite) in the presence of a strong acid such as hydrochloric, phosphoric, etc. This reaction is carried out in the presence of either water and/or a non-aqueous solvent such as dimethylformamide, tetrahydrofuran, dioxane, chloroform, methylene chloride, etc., at a temperature between about -40° C. and +20° C. Alternatively, the peptide may be removed from the resin by treatment with a lower alcohol such as methanol in the presence of an organic base such as triethylamine, thus resulting in the formation of the corresponding lower alcohol ester of the linear peptide. The resulting ester may be converted to the hydrazide which may then be cyclized, via the azide, to the desired cyclic peptide. The preferred method for cleaving the peptide from the resin in the present invention is the use of hydrazine.

As reference Table II will show, one preferred overall procedure for preparing the desired cyclic peptides of the present invention involves the stepwise synthesis of the linear peptide on a solid phase resin. More specifically, in the process for preparing: ##STR5## the carboxyl end of the N-blocked amino acid proline(N-Me-Lys peptide) or phenylalanine (N-Me-Phe peptide) is bound covalently to an insoluble polymeric resin support as the carboxylic acid ester of the resin-bonded benzyl chloride. The amino group of Phe is protected by the BOC group. After the attachment of the Phe is completed on the resin, the protecting group BOC is removed by treatment with TFA in CH₂ Cl₂. The subsequent amino acids are attached, in the form of BOC-amino acid, using DCCI as the condensing agent or an active ester such as ONp. N-Me-Lys is introduced as the N.sup.α -FMOC derivative. After the desired linear peptide has been prepared, the N-terminal amino group is selectively deblocked and the peptide is removed from the resin by treatment with hydrazine. The resulting linear peptide hydrazide with the N-terminal amino group deblocked having the amino acid sequence: ##STR6## is treated with isoamyl nitrite in acid pH to form the corresponding azide. The azide solution is diluted with solvent and neutralized with an organic base. The linear peptide cyclizes to form: ##STR7## During the cyclization the "pH" is checked and maintained at neutral by the addition of organic base. The "pH" in organic solvent is determined by the application of an aliquot of the solution to moistened narrow range pH paper.

After the linear peptide is cyclized, the protective groups, 2-Cl-CBZ and OBzl, are removed by treatment with HF in the presence of anisole. The crude cyclic peptide obtained is purified chromatographically, preferably with column chromatography on silica gel. The elution is generally an organic solvent or mixtures thereof which is selected by analyzing aliquots of the material using thin layer chromatography.

                                      TABLE II                                     __________________________________________________________________________     The reaction schemes for the preparation of                                    two of the cyclic hexapeptides of this invention are                           outlined in the following series of reactions:                                 __________________________________________________________________________     Reaction scheme for preparing:                                                  ##STR8##                                                                       ##STR9##                                                                       ##STR10##                                                                      ##STR11##                                                                      ##STR12##                                                                      ##STR13##                                                                      ##STR14##                                                                      ##STR15##                                                                     Reaction scheme for preparing:                                                  ##STR16##                                                                      ##STR17##                                                                      ##STR18##                                                                      ##STR19##                                                                      ##STR20##                                                                      ##STR21##                                                                      ##STR22##                                                                      ##STR23##                                                                     __________________________________________________________________________

The following Examples are given to illustrate the methods used to carry out the present invention. It is to be understood that these Examples are given for purposes of illustration and not limitation.

EXAMPLE 1 Preparation of D-Trp-(-2-Cl-CBZ)Lys-(OBZl)Thr-N-Me-Phe-Pro-Phe-OCH₂ φ-resin

Chloromethyl resin (2% cross-linked Merrifield resin), 862.0 g (2.37 moles), having 2.75 meq. chlorine/g, and 607.0 g (2.37 moles), 1 equivalent) of BOC-Phe were added to 4320 ml of peroxide-free tetrahydrofuran. The mixture was stirred in an oil bath at 80° C. bath temperature for 45 minutes. Triethylamine, 310.0 ml, was added and the reaction mixture stirred at 80° C. bath temperature for 70 hours, cooled to 25° C. and transferred to a stirred solid phase reaction column with 2000 ml of tetrahydrofuran. After removal of the solvent, the resin was washed using the stirred column with:

3×2000 ml of tetrahydrofuran

4×5170 ml of ethanol

1×5170 ml of acetic acid

3×5170 ml of water

3×5170 ml of methanol

3×5170 ml of chloroform

The BOC-Phe-O-CH₂ φ-resin was dried in vacuo at 25° C. for 16 hours, giving 1203 g of BOC-Phe-O-CH₂ φ-resin containing 1.2 mmole of phenylalanine/g of resin.

BOC-Phe-O-CH₂ φ-resin (1.67 g; 2.0 mmole) was carried through the procedures in Tables III and IV using 2 deblockings (2 minutes and 25 minutes) with 25% TFA in methylene chloride and 2.5 equivalents of BOC-amino acid in the required sequence until the desired BOC-hexapeptide-O-CH₂ φ-resin was obtained.

DCCI was used as the sole coupling agent in every step.

The coupling of each amino acid proceeded smoothly. Best yields were obtained when the coupling was repeated in each step. When the coupling was repeated, the inital two chloroform washes, the deblocking step and the succeeding three chloroform washes were all omitted and replaced by a single chloroform wash.

The coupling reactions were carried out in methylene chloride, freshly degassed DMF or a mixture of these two solvents. The N-terminal amino group was blocked with a BOC group in each case; the hydroxy group of Thr was blocked with Bzl and the ε-amino group of Lys with 2-Cl-CBZ.

When the desired BOC-hexapeptide-O-CH₂ φ-resin was obtained, the N-terminal BOC group was removed by the terminal deblocking procedure set forth in Table V.

                                      TABLE III                                    __________________________________________________________________________     Solvent or 25% TFA                                                                              CHCl.sub.3 (3)                                                                      Net.sub.3 -                                                                         CHCl.sub.3 (3)                                                                       BOC AA                                                                               0.5 M DCCI                                                                            DMF(1)                           reagent                                                                               CHCl.sub.3                                                                         in         CH.sub.2 Cl.sub.2                                                                   CH.sub.2 Cl.sub.2 (3)                                                                in    in     MeOH(1)                          (number of                                                                            (2) CH.sub.2 Cl.sub.2                                                                         (1:9)      CH.sub.2 Cl.sub.2                                                                    CH.sub.2 Cl.sub.2                                                                     DMF(1)                           treatments                       DMF or a     MeOH(1)                          or washes) (2)        (2)        mixture of   CHCl.sub.3 (2)                                                    both                                          Vol. in ml.                                                                           40  20    40   40   40    25    10     40                               Time in min.                                                                           5  2 and 25                                                                              2   5 and 5                                                                              2     5     5      2                                                                      coupling 30                             __________________________________________________________________________

                  TABLE IV                                                         ______________________________________                                         Protected Amino Acid      Solvent Ml                                           ______________________________________                                         BOC--Pro (1.08 g)         20 ml CH.sub.2 Cl.sub.2                              Recouple                                                                       BOC--N--Me--Phe (1.40 g)  20 ml CH.sub.2 Cl.sub.2                              Recouple                                                                       BOC(OBzl)Thr (1.55 g)     20 ml CH.sub.2 Cl.sub.2                              Recouple                                                                       BOC--(2-Cl--CBZ)Lys (2.07 g)                                                                             20 ml CH.sub.2 Cl.sub.2                              Recouple                                                                       BOC--D-Trp (1.52 g)       15 ml CH.sub.2 CH.sub.2,                             Recouple                  5 ml DMF -                                           II In a similar manner the other cyclic hexapeptide is                         prepared using Pro as the first amino acid                                     BOC--Phe (1.32 g)         25 ml CH.sub.2 Cl.sub.2                              Recouple                                                                       BOC(OBzl)Thr (1.55 g)     25 ml CH.sub.2 Cl.sub.2                              Recouple                                                                       FMOC--(2-Cl--CBZ)N--Me--Lys (2.75 g)                                                                     25 ml CH.sub.2 Cl.sub.2                              Recouple                                                                       BOC--D-Trp (1.52 g)       20 ml CH.sub.2 Cl.sub.2,                             Recouple                  5 ml DMF                                             BOC--Phe (1.32 g)         25 ml CH.sub.2 Cl.sub.2                              Recouple                                                                       ______________________________________                                    

                  TABLE V                                                          ______________________________________                                         Solvent  CHCl.sub.3 (1)                                                                           25% TFA in        MeOH(2)                                   or reagent         CH.sub.2 Cl.sub.2 + 1%                                                                     CHCl.sub.3                                                                           CH.sub.2 Cl.sub.2 (1)                     (number of         Ethanedithiol                                                                              (3)   MeOH(2)                                   treatments         (2)               CH.sub.2 Cl.sub.2 (2)                     or washes)                                                                     Vol. in ml.                                                                             40        40          40    40                                        Time in   5        2 and 25     2     2                                        minutes                                                                        ______________________________________                                    

After the procedures of Tables III, IV an V were completed, the blocked hexapeptide-OCH₂ φ-resin is dried overnight and weighs 3.8 g.

EXAMPLE 2 Preparation of D-Trp-(-2-Cl-CBZ)Lys-(OBZl) Thr-N-Me-Phe-Pro-Phe-NHNH₂

The resin from Example 1 was combined with 40 ml of a 1:1 mixture of methanol and hydrazine and stirred at room temperature for 1 hour. The insoluble resin was removed by filtration and the solution is evaporated to remove the methanol and hydrazine. Reconcentration from methanol twice and water, once, followed by trituration with water, gave a solid. Filtration, washing with water and drying, gave 2.27 g of a solid.

EXAMPLE 3 Preparation of D-Trp-(-2-Cl-CBZ)Lys-(OBZl) Thr-N-Me-Phe-Pro-Phe-N₃

The solid from Example 2 was combined with 22 ml of degassed dimethylformamide under a blanket of nitrogen and cooled to -10° C., and 5 equivalents of 4.52M hydrogen chloride in tetrahydrofuran (2.2 ml) were added. The solution was cooled to -25° C. and 5 ml of a 1:19 mixture of isoamyl nitrite in dimethylformamide was added in portions until a positive starch/KI test was obtained. The completion of the reaction is followed by thin layer chromatography and the disappearance of the hydrazide starting material.

EXAMPLE 4 Preparation of Cyclo(D-Trp-(2-Cl-CBZ)Lys-(OBZl)-Thr-N-Me-Phe-Pro-Phe)

The azide compound of Example 3 was added to 600 ml of degassed dimethylformamide, precooled to -25°, the pH adjusted to 8, with triethylamine, and the reaction mixture placed in the freezer overnight. The pH was readjusted to 8 if necessary after about 14 hours and the mixture stored for 16 hours at -20° C. and 16 hours at 5° C. Thin layer chromatography indicated that the reaction was completed. The mixture was concentrated to dryness, dissolved in 150 ml of a 3:1 dimethylformamide/water mixture and treated with a mixed bed anion-cation exchange resin for 1 hour. The mixture was filtered and concentrated to dryness in vacuo to an oil, and the residue is triturated with methanol, affording 1.5 g of a white solid.

EXAMPLE 5 Preparation of Cyclo(D-Trp-Lys-Thr-N-Me-Phe-Pro-Phe)

1.5 G (1.8 mmoles) of the protected cyclic hexapeptide of Example 4 was combined in a teflon lined chamberwith 2 ml of anisole. The chamber was then evacuated and filled with liquid hydrogen fluoride at the temperature of the dry ice/acetone bath. The temperature was raised to 0° C. and stirring continued for 1 hour. The hydrogen fluoride was allowed to evaporate and the residue placed in vacuo until a slurry was formed. The slurry was triturated with ethyl acetate and filtered affording 1.0 g of a fine powder. The powder was placed on G-25 Sephadex eluted with a 50% aqueous acetic acid, eluting with 9 ml fractions. Fractions 53-60 were combined and concentrated. The product was freeze-dried affording 200 mg of solid material.

EXAMPLE 6 Preparation of Phe-D-Trp-(-2-Cl-CBZ)-N-Me-Lys-(OBZl)Thr-Phe-Pro-NHNH₂

The resin from Example 1 (1.5 mmoles) was combined with 40 ml of 1:1 mixture of methanol and hydrazine and stirred at room temperature for 1 hour. The insoluble resin was removed by filtration and the solution was evaporated to remove the methanol and hydrazine. Reconcentration from methanol twice and water followed by trituration with water gave a solid. Filtration, washing with water and drying gave 1.05 g of solid.

EXAMPLE 7 Preparation of Phe-D-Trp-(2-Cl-CBZ)-N-Me-Lys-(OBZl)Tnr-Phe-Pro-N₃

The solid from Example 6 was combined with 20 ml of degassed dimethylformamide under a blanket of nitrogen and cooled to -10° C., and 5 equivalents of 5.5 M hydrogen chloride in tetrahydrofuran (17 ml) was added. The solution was cooled to -25° C. and 5 ml of a 1:19 mixture of isoamyl nitrite in dimethylformamide was added. The completion of the reaction was followed by thin layer chromatography and the disappearance of the hydrazide starting material.

EXAMPLE 8 Preparation of Cyclo(D-Trp-(2-Cl-CBZ)N-Me-Lys-(OBZl)-Thr-Phe-Pro-Phe)

The azide compound of Example 7 was added to 600 ml of degassed dimethylformamide, precooled to -25° C., the pH adjusted to 8, and the reaction mixture placed in the freezer overnight. The pH was readjusted to 8 if necessary after about 14 hours and the mixture stored for 16 hours at -20° C. and 16 hours at 5° C. Thin layer chromatography indicates that the reaction was completed. The mixture was concentrated to dryness, dissolved in 150 ml of 3:1 dimethylformamide/water mixture and treated with a mixed bed anion-cation exchange resin for 2 hours. The mixture was filtered and concentrated to dryness in vacuo, and triturated with water to a solid. Filtration and drying gave 908 mg of solid.

EXAMPLE 9 Preparation of Cyclo(D-Trp-N-Me-Lys-Thr-Phe-Pro-Phe)

908 Mg of the protected cyclic hexapeptide of Example 8 was combined in a teflon lined chamber with 3 ml of anisole. The chamber was then evacuated and filled with liquid hydrogen fluoride at the temperature of the dry ice/acetone bath. The temperature was raised to 0° C. and stirring continued for 1 hour. The hydrogen fluoride was allowed to evaporate and the residue placed in vacuo until a slurry is formed. The slurry was treated with ethyl acetate and filtered affording 562 mg of a fine powder. This material was purified by flash chromatography on silica gel eluted with an 80:20:2 mixture of chloroform, methanol and concentrated aqueous ammonia, affording 250 mg of product.

Following the above procedure, and by modifying only the selection and order of amino acids in the process of Example 1, there are prepared other cyclic hexapeptides of this invention.

The instant cyclic hexapeptide analogs of somatostatin are tested and compared with the effects of somatostatin in an in vitro test for the inhibition of growth hormone. The test is described as follows:

"Rat pituitaries were isolated according to the procedures of Vale and Grant "In vitro Pituitary Hormone Secretion Assay for Hypophysiotropic Substances" in Methods in Enzymology, Vol. XXXVII, eds. O'Malley, B.W. and Hardman, J.G. (Academic Press, Inc., New York) pp. 5-93 (1975).

After 4 days in culture, the cells were washed and incubated for 4 hours in Dulbecco-modified Eagle's medium in the presence or absence of graded doses of each analog or somatostatin. The medium was then collected for subsequent growth hormone determinatin by a double antibody radioimmunoassay for rat growth hormone."

Analogs of somatostatin were compared to somatostatin in their ability to decrease the levels of portal vein glucagon and insulin in anesthetized rates. Male Sprague-Dawley rats (Charles River CD) weighing 160-200 g were anesthetized with urethane (150 mg/100 g of body weight; Aldrich). Saline or peptides were administered via the external jugular vein. After 5 minutes, the portal vein was exposed, and blood was collected via syringe containing 3 mg of EDTA and placed in chilled tubes containing 100 μl of Trasylol (FBA Pharmaceuticals) for subsequent hormone analysis. Plasma levels of glucagon were determined by the method of Faloona and Unger, Methods of Hormone Radioimmunoassay, Jaffe and Behrman (Eds), Academic Press, New York, Vol. II, pp. 257-527 (1976), utilizing glucagon antisera 30K obtained from R. Unger (Dallas, TX). Plasma levels of insulin were determined by a modification of the procedure of Herbert et al., J. Clin. Endocrinol. Metab., 25, 1375-1384 (1965).

The test results for some of the compounds of this invention are recorded below with the results for somatostatin listed first and given the arbitrary value of 1. The results for the instant compounds are given as multiples or fractions of the effect of somatostatin. The numbers in parentheses are the fiducial limits for the number preceding. The first two of the instant compounds listed are the compounds prepared in Examples 1-5 and 6-9 respectively. The compounds are written slightly different, however, to conform to the order of the amino acids found in somatostatin.

    ______________________________________                                         Activity of Cyclichexapeptide Analogs of Somatostatin                                         Growth                                                                         Hormone              Gluca-                                                    Release    Insulin   gon                                        Compound       Inhibition Inhibition                                                                               Inhibition                                 ______________________________________                                         Somatostatin    1          1         1                                         Cyclo(Pro--Phe--                                                                              0.53(0.34,0.82)                                                                           1.1(0.6,1.7)                                                                             2.0(1.1,                                   D-Trp--Lys--                        4.0)                                       Thr--N--Me--Phe)                                                               Cyclo(Pro--Phe--                                                                              1.6(0.7,3.4))                                                                             0.5(0.1,1.8)                                                                             Active                                     D-TrP--L-N--Me--Lys--                                                          The--Phe)                                                                      Cyclo(Pro--Phe--                                                                              1.5(1.2,1.9)                                                                              0.4(0.01,1.9)                                                                            0.4(0.1,                                   D-Trp--D-N--Me--Lys--               0.7)                                       Thr--Phe)                                                                      Cyclo(Pro--Phe--                                                                              0.1(0.09,0.2)                                                                             0.3(0.1,0.7)                                                                             0.2(0.1,                                   L-Trp--L-N--Me--Lys--               0.7)                                       Thr--Phe)                                                                      Cyclo(Pro--Phe--                                                                              0.05(0.04,0.07)                                                                           0.07       --                                        L-Trp--D-N--Me--Lys--                                                          Thr--Phe)                                                                      ______________________________________                                    

The effects of the instant cyclichexapeptide analogs of somatostatin are determined by the following procedure.

Compounds were tested for their ability to inhibit pentagastrin evoked gastric secretion in the chronic fistula dog. Female beagle dogs with a chronic gastric fistula were given pentagastrin (2.5 μg./kg./hour, i.v. from -60 to 120 min.) and gastric outputs were collected via the fistula cannula. Samples were analyzed at 30 minute intervals for volume (Ml.) and titratable acid (mEq/L) (Titration to pH 7 with 0.01N NaOH); total acid output (mEq) was calculated as the production of output volume and acid concentration. Test compounds were infused at a constant rate from 0 to 60 minutes. Data have been expressed as percent change of total acid output relative to a placebo trial in the same animals.

In the following data, the results for somatostatin are given first for comparison purposes.

    __________________________________________________________________________     Effects of Cyclichexapeptide analogs of Somatostatin on Gastric                Secretion                                                                      (Dose 0.8 μg/ml./min.-infusion 0-60 min.)                                                 Gastric Secretion & Inhibition                                                 Vol.           Acid Concentration                                Compound      0-30                                                                              30-60                                                                              60-90                                                                              90-120                                                                             0-30                                                                              30-60                                                                              60-90                                                                              90-120                                 __________________________________________________________________________     Somatostatin  85 97  81  37  16 77  61  14                                     Cyclo(Pro--Phe--D-Trp--                                                                      78 82  61  60   9 49  43  32                                     N--Me--Lys--Thr--Phe)                                                          Cyclo(Pro--Phe--D-Trp--                                                                      82 89  49  60  44 51   6   0                                     N--Me--D-Lys--The--Phe)                                                        __________________________________________________________________________ 

What is claimed is:
 1. A compound having the formula: ##STR24## wherein X is (CH₂)_(n) wherein n is 0, 1 or 2 or sulfur;Y is (CH₂)_(m) wherein m is 0, 1 or 2 or sulfur such that the sulfur may be in any position along the chain; R₁ and R₂ are independently lower alkyl, benzyl, substituted benzyl wherein the substituent may be one or two of loweralkyl, halogen, hydroxy, amino, nitro or loweralkoxy; and loweralkyl substituted with hydroxy, carboxy, amino, guanidino, or a 5- or 6-membered heterocyclic ring; R₃ is 3-indolylmethyl or substituted 3-indolylmethyl wherein the substituent may be loweralkyl, loweralkoxy or halogen; R₄ is loweralkyl, hydroxyloweralkyl, benzyl, carboxyloweralkyl, aminoloweralkyl or substituted hydroxy benzyl wherein the substituent may be loweralkyl, loweralkoxy, hydroxy, halogen, amino or nitro; R₅ is loweralkyl, benzyl, or substituted benzyl wherein the substituent is loweralkyl, loweralkoxy, hydroxy, halogen, amino or nitro; and R₆ and R₇ are independently hydrogen or straight chain loweralkyl provided at least one of R₆ and R₇ is loweralkyl.
 2. A compound of claim 1 wherein X are (CH₂)_(n) and n is 1;Y, R₁, and R₂ are as defined in claim 1; R₃ is 3-indolylmetnyl or substituted indolylmethyl wherein the substituent is methoxy or fluoro; R₄ is methyl, ethyl, isopropyl, hydroxymethyl or hydroxyethyl; R₅ is methyl or benzyl; and one or both of R₆ and R₇ are methyl.
 3. A compound of claim 2 wherein:X and Y are methylene; R₁ and R₂ are as defined in claim 1; R₃ is 3-indolylmethyl; R₄ is hydroxyethyl; R₅ is methyl or benzyl; and one or both of R₆ and R₇ are methyl.
 4. The compound of claim 2 which is cyclo (Pro-Phe-D-Trp-L-N-Me-Lys-Thr-Phe).
 5. The compound of claim 2 which is cyclo (Pro-Phe-D-Trp-D-N-Me-Lys-Thr-Phe).
 6. The compound of claim 2 which is cyclo (Pro-Phe-L-Trp-L-N-Me-Lys-Thr-Phe).
 7. The compound of claim 2 which is cyclo (Pro-Phe-L-Trp-D-N-Me-Lys-Thr-Phe).
 8. The compound of claim 2 which is cyclo (Pro-Phe-D-Trp-Lys-Thr-N-Me-Phe).
 9. A method for inhibiting the release of growth hormone which comprises administering to an animal an effective amount of a cyclic hexapeptide of claim
 1. 10. A pharmaceutical composition for the treatment of diabetes comprising a therapeutically effective amount of the cyclic hexapeptide of claim 1 or the non-toxic acid addition salts thereof in a pharmaceutically acceptable liquid or solid carrier. 